Na+ absorption by airway epithelia influences the rate of mucociliary clearance (MCC) by adjusting the depth of airway surface liquid (ASL). Excessive Na+ absorption decreases, and inhibited Na+ absorption stimulates MCC. The amiloride sensitive epithelial Na+ channel (ENaC) in the apical membrane is the rate-limiting step in Na+ absorption. Although ENaC is expressed in many tissues and is well studied, most investigations have focused on hormonal stimulation of ENaC in salt conserving epithelia. Na+ absorption in airways plays no role in salt conservation and is unaffected by hormones that stimulate ENaC. Instead, signals present in ASL, such as nucleotides, typically increase the depth of ASL and the rate of MCC by inhibiting Na+ absorption. This effect predicts signaling pathways that link local stimuli to ENaC activity, but knowledge about these transduction mechanisms is limited. Each of ENaC's three homologous subunits contains a highly conserved gating domain in its cytosolic amino terminus. We hypothesize that inhibitory stimuli present in ASL decrease Na+ absorption through pathways that interact with ENaC amino termini to decrease ENaC open probability. We will test this hypothesis for the case of nucleotides in ASL. Nucleotides are known to activate the beta isozymes of phospholipase C (PLCbeta). In Aim 1, we will test the role of PLCbeta activity in the regulation of ENaC in the apical membrane of airway epithelial cells and we will identify the specific PLCbeta isozyme(s) in the apical membrane and determine PLC protein-binding partners. In Aim 2 we will test the hypothesis that the PLC substrate, 4,5 phosphoinositide phosphate (PIP2), binds to the ENaC amino termini and stabilizes the ENaC open conformation by identifying the specific residues that interact with PIP2 and other anionic phospholipids. Finally, in Aim 3 we will use a proteomic approach to identify proteins that interact with the amino termini of ENaC. We will determine if these interactions regulate ENaC gating and are affected by PIP2 binding to the ENaC amino termini.